Method for preparing low endotoxin chitosan

Abstract

The present invention relates to a method of preparing low endotoxin alkali chitosan, and to a method of preparing low endotoxin neutral chitosan, chitosan salts and chitosan derivatives, and to the products of these methods. The method comprises contacting chitosan with an alkali solution to form a mixture, and allowing the mixture to stand for at least about 12 hours. Other useful chitosan-based products can be prepared using the low endotoxin alkali chitosan.

Description

[0001] This application is a divisional application of the application for patent filed on 25 March 2013 under the number 201380026792.3 entitled "Process for the preparation of low endotoxin chitosan". TECHNICAL FIELD

[0002] The present invention relates to a process for the preparation of low endotoxin basic chitosan, to a process for the preparation of low endotoxin neutral chitosan, chitosan salts and chitosan derivatives, and to the products of these processes. BACKGROUND

[0003] Chitosan is particularly used for the preparation of hemostatic materials for use in the control of bleeding.

[0004] Chitosan is a derivative of solid waste from the processing of crustaceans and can be extracted from fungal cultures. Chitosan is a water-insoluble cationic polymeric substance. Before chitosan is used in hemostatic materials, it is usually first converted into a water-soluble salt. In this way, the chitosan salt is soluble in blood to form a gel that stops the flow of blood.

[0005] Since chitosan is easily broken down in the body, chitosan salts are ideally suited for the applications described herein. Chitosan is converted by lysozyme into glucosamine and is therefore naturally excreted from the body. It is not necessary to remove chitosan from the body. In addition, chitosan salts show mild antibacterial properties, so their use reduces the risk of infection.

[0006] In order to utilize chitosan in the preparation of hemostatic materials suitable for the control of bleeding, it is necessary to ensure that the chitosan has a sufficiently low endotoxin concentration.

[0007] Endotoxins are lipopolysaccharides present on the outer membrane surface of Gram-negative bacteria. Endotoxins are highly toxic to mammals, in particular to humans, and are notoriously difficult to remove from materials. When released into the bloodstream or other tissues where they are not normally found, endotoxins can become pyrogenic. Therefore, endotoxins must be removed from pharmaceutically acceptable products.

[0008] The process of removing or destroying pyrogens, in particular endotoxins, is known as "depyrogenation". Techniques for depyrogenating materials containing endotoxins include ion exchange chromatography, ultrafiltration, distillation and various chemical methods directed at destroying endotoxins.

[0009] WO2008063503 relates to a process for removing endotoxins from chitosan, said process comprising the steps of:

[0010] a) using sterile depyrogenated equipment and materials in a sterile environment;

[0011] b) swelling the chitosan containing endotoxins for up to 24 hours;

[0012] c) Dissolve 1 kg / 25 L to 1.5 kg / 25 L chitosan in 0.01 M to 4.0 M hydroxide base;

[0013] d) Continuously stir the resulting chitosan base solution;

[0014] e) Heat the solution between 60-100 °C for 45 minutes to 4 hours with stirring;

[0015] f) Rinse the solution with up to 10x volume of ultrapure endotoxin-free water;

[0016] g) Neutralize the solution to a pH between 6.8 and 7.5;

[0017] h) Form an ultrapure low endotoxin chitosan slurry and transfer to an endotoxin-free closed system;

[0018] i) Remove excess water from the slurry.

[0019] This is a complex and costly process, requiring, inter alia, sterile equipment, and requiring rinsing the solution with 10x volume of endotoxin-free water.

[0020] US2006293509 relates to a method of preparing water-soluble chitosan having low endotoxin by:

[0021] (a) contacting water-insoluble chitosan with a basic solution for a first period of time greater than 1 hour;

[0022] (b) optionally rinsing the water-insoluble chitosan with endotoxin-free water to remove residual basic solution;

[0023] (c) partially acetylating the water-insoluble chitosan in a reaction solution comprising a phase transfer agent;

[0024] (d) dissolving the partially acetylated water-soluble chitosan in an aqueous solution comprising a surfactant and having a pH of about 7.0 to about 7.4;

[0025] (e) adding a water-miscible solvent to the aqueous solution and further adjusting the pH of the aqueous solution to a pH of at least 8.0 to precipitate the water-soluble chitosan having a low endotoxin content from the aqueous solution / water-miscible solvent mixture; and

[0026] (f) optionally washing in a non-solvent, e.g., isopropanol.

[0027] However, this process is complex and costly, and desirably involves the use of large amounts of endotoxin-free water or other liquids. This process also requires the use of a phase transfer agent, and is carried out over several hours.

[0028] TW593342 relates to a method of reducing endotoxin in chitosan by: (a) dissolving chitosan in a basic solution;

[0029] (a) dissolving chitosan comprising endotoxin in an aqueous solution;

[0030] (b) contacting the aqueous solution with a surfactant to form an insoluble solid and an aqueous solution having a reduced endotoxin content;

[0031] (c) separating the solid from the aqueous solution using a solid / liquid separation device.

[0032] However, this process requires a surfactant to react with the dissolved chitosan to produce an insoluble solid. The resulting solid is a mixture of chitosan and surfactant or a reaction product between chitosan and surfactant. SUMMARY

[0033] The present invention is directed to alleviating the aforementioned difficulties.

[0034] According to a first aspect of the present invention, there is provided a method of preparing low endotoxin alkali chitosan, the method comprising the steps of:

[0035] (a) contacting chitosan with an alkali solution to form a mixture; and

[0036] (b) allowing the mixture to stand for at least about 12 hours.

[0037] According to another aspect of the present invention, there is provided a method of preparing low endotoxin alkali chitosan, the method comprising the steps of:

[0038] (a) contacting chitosan with an alkali solution to form a mixture;

[0039] (b) allowing the mixture to stand for at least about 12 hours; and

[0040] (c) drying the mixture.

[0041] The method of the present invention provides an efficient way of obtaining alkali chitosan having a low endotoxin concentration. The method advantageously does not require a washing step, a rinsing step, the use of a surfactant or a phase transfer agent, sterile equipment and / or the use of endotoxin-free water. In addition, no special air filtration or sterile conditions are required. The method of the present invention preferably does not include a step of acetylating the chitosan.

[0042] The term "alkali solution" as used herein refers to a solution having a pH value greater than pH 7.5.

[0043] Since the molecular weight of endotoxin can vary significantly, the endotoxin concentration is measured in endotoxin units (EU) per gram of material. The measurement of endotoxin concentration is a quantification of the level of endotoxin relative to a specific amount of reference endotoxin.

[0044] For example, in the present application, the endotoxin concentration is measured in endotoxin units (EU) per gram of chitosan. As used herein, the term "low endotoxin" refers to an endotoxin concentration of less than 100 endotoxin units (EU) per gram of chitosan.

[0045] Thus, the process of the present application is suitable for producing basic chitosan having an endotoxin concentration of less than 100 EU / g.

[0046] Preferably, the resulting basic chitosan has an endotoxin concentration of less than 50 EU / g, more preferably less than 20 EU / g, even more preferably less than 15 EU / g, and most preferably less than 10 EU / g.

[0047] It has been found that a low concentration of the basic solution is preferred in the process of the present application. The concentration of the basic solution used in the process can be from about 0.01 M to about 1 M. Preferably, the concentration of the basic solution is less than 1 M. Preferably, the concentration of the basic solution is from about 0.02 M to 0.2 M, and even more preferably, the concentration of the basic solution is from about 0.04 M to 0.06 M, typically 0.05 M. The concentration of the basic solution can be about 0.01 M, 0.05 M, 0.10 M, 0.15 M, 0.20 M, 0.25 M, 0.30 M, 0.35 M, 0.40 M, 0.45 M, 0.50 M, 0.55 M, 0.60 M, 0.65 M, 0.70 M, 0.75 M, 0.80 M, 0.85 M, 0.90 M, or 0.95 M. Good results have been observed with a concentration of 0.1 M basic solution.

[0048] In some embodiments, the amount of basic solution to chitosan can be from about 1 part chitosan to about 10 parts basic solution to about 10 parts chitosan to about 1 part basic solution. Preferably, the amount of basic solution to chitosan is about 1 part basic solution to about 2 parts chitosan, and more preferably, about 1 part basic solution to about 1 part chitosan.

[0049] The basic solution can comprise an alkali or alkaline earth component selected from the group consisting of metal hydroxides, metal carbonates, metal bisulfites, metal persilicates, conjugate bases, and ammonium hydroxide, alone or in combination.

[0050] Suitable metals include sodium, potassium, calcium, or magnesium.

[0051] Preferably, the alkali component is sodium hydroxide, potassium hydroxide, or sodium carbonate. Typically, sodium hydroxide is used.

[0052] The basic solution can be contacted with the chitosan by any suitable method known in the art. For example, the basic solution can be sprayed onto the chitosan, or the chitosan can be mixed with the basic solution. Preferably, the alkali contacted chitosan is uniformly distributed.

[0053] Preferably, chitosan is mixed with an alkaline solution. At low molecular weights, chitosan is completely or partially soluble in the alkaline solution. The chitosan can be mixed with the alkaline solution for up to about 30 minutes, more preferably about 10 minutes. In some embodiments, the chitosan can be mixed with the alkaline solution for more than 30 minutes. In some embodiments, the duration of step (b) of intermittent stirring of the mixture of chitosan and alkaline solution can be adjusted.

[0054] The mixture of chitosan and alkaline solution is held for a period of time during which the endotoxin is destroyed by the alkali. The mixture of chitosan and alkaline solution is held for at least about 12 hours. It has been found that the longer the mixture of chitosan and alkaline solution is held, the lower the endotoxin concentration of the resulting alkaline chitosan. Suitable low endotoxin concentrations have been observed when the mixture has been held for about 12 hours. Another advantage of the method of the present invention is that the mixture can be held without the need for continuous mixing of chitosan and alkaline solution.

[0055] In some embodiments, the mixture may be maintained for at least about 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 hours.

[0056] It is preferable to keep the mixture for at least 48 hours.

[0057] In some implementations, the mixture may be maintained for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 days or longer.

[0058] In some embodiments, the mixture is kept for about 2 to 4 weeks (or 14 to 30 days) or longer. Preferably, the mixture is kept for between 24 hours and 70 days, more preferably between 7 days and 35 days, and most preferably between 14 days and 21 days.

[0059] Good results have been observed by contacting chitosan with a 0.1M sodium hydroxide solution and keeping the mixture in contact for about 12 to 16 days, preferably about 14 days.

[0060] The mixture can be maintained at room temperature and room pressure. Room temperature and room pressure refer to a temperature of approximately 20-25°C and a pressure of approximately 1 atmosphere (atm). Advantageously, the mixture does not need to be maintained in a sterile environment.

[0061] The mixture is preferably stored in a clean container. It may also be stored under an inert atmosphere.

[0062] The mixture may further contain preservatives. Advantageously, preservatives can eliminate the risk of microbial growth, which may occur, for example, when the mixture is stored for a long period of time. Preservatives can be any biocompatible preservative suitable for alkaline environments. Suitable preservatives include silver ions, zinc ions, chlorohexadine, or combinations thereof.

[0063] The method of the present invention may or may not include a drying step. The drying step can be performed by any conventional drying method known in the art. Preferably, the drying step is performed in an oven or by filtering through an air dryer. Likewise, the drying step does not require specialized aseptic equipment.

[0064] It has been found that once the mixture has been dried in the drying step, the endotoxin level of the mixture no longer increases significantly. This is advantageous for further processing of the mixture.

[0065] Therefore, the present invention provides a low-endotoxin basic chitosan with an endotoxin concentration of less than 100 EU / g. Low-endotoxin basic chitosan can be water-insoluble. At low molecular weights, low-endotoxin basic chitosan can exhibit some water solubility.

[0066] According to another aspect of the invention, a low-endotoxin basic chitosan is provided that can be obtained by the methods described herein.

[0067] According to another aspect of the invention, an alkaline chitosan having an endotoxin concentration of less than 100 EU / g is provided.

[0068] The alkaline chitosan preferably has an endotoxin concentration of less than 50 EU / g, more preferably less than 20 EU / g, even more preferably less than 15 EU / g, and most preferably less than 10 EU / g.

[0069] Low-endotoxin basic chitosan can be used to prepare other chitosan products, such as derivatives or copolymers, or to prepare low molecular weight chitosan or chitosan oligosaccharides. Low-endotoxin basic chitosan can also be used as a raw material to prepare other forms of chitosan or derivatives or copolymers, such as chitosan-based fibers, fabrics, coatings, films, gels, solutions, sheets or foams.

[0070] Specifically, low-endotoxin basic chitosan can be used to prepare other useful chitosan products with low endotoxin concentrations, including neutral chitosan and chitosan salts and other chitosan derivatives, such as carboxymethyl chitosan, hydroxyethyl chitosan, acyl chitosan, alkyl chitosan, sulfonyl chitosan, phosphorylated chitosan, alkylidene chitosan, metal chelates, chlorinated chitosan, lactic acid chitosan, acetate chitosan, malic acid chitosan, and gluconate chitosan.

[0071] Therefore, according to another aspect of the present invention, a method for preparing low endotoxin neutral chitosan, chitosan salt, or chitosan derivative is provided, the method comprising the step of contacting an acid with an alkaline chitosan prepared by the method described herein.

[0072] This method can provide medical-grade neutral chitosan, chitosan salts, or other chitosan derivatives with low concentrations of endotoxin.

[0073] In the method of the first aspect of the present invention, the step of contacting the alkaline chitosan with the acid can be performed before the drying step.

[0074] The acid can be brought into contact with the basic chitosan by any suitable method known in the art. For example, the acid can be sprayed onto the basic chitosan, or the basic chitosan can be mixed with the acid.

[0075] It is preferable to mix alkaline chitosan with acid.

[0076] In this document, neutral chitosan refers to chitosan having a pH value between about 6.5 and about 7.5, preferably about 7.

[0077] Therefore, to prepare neutral chitosan, basic chitosan can be mixed with an acid of suitable volume and concentration to generate a neutral solution with a pH between 6.5 and 7.5. The volume and / or concentration of acid required to neutralize basic chitosan will depend on the pH of the basic chitosan.

[0078] Alternatively, to prepare chitosan salts or chitosan derivatives, basic chitosan can be mixed with an acid of a volume and concentration exceeding that required to provide neutral chitosan.

[0079] Suitable acids for use in this invention may be selected from the following acids, alone or in combination: organic acids, carboxylic acids, fatty acids, amino acids, Lewis acids, monoprotic acids, diprotic acids, polyprotic acids, nucleic acids, and inorganic acids.

[0080] Suitable organic acids can be selected from the following organic acids alone or in combination: acetic acid, tartaric acid, citric acid, ascorbic acid, acetylsalicylic acid, gluconic acid, and lactic acid.

[0081] Suitable fatty acids can be selected from the following fatty acids, alone or in combination: myristicinic acid, palmitoleic acid, cis-6-hexadecenoic acid, oleic acid, trans-oleic acid, 11-octadecenoic acid, linoleic acid, trans-linolenic acid, alpha-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, docosahexaenoic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, and sorbic acid. Acids, lignoceric acid, ceric acid.

[0082] Suitable amino acids can be selected from the following amino acids alone or in combination: histidine, lysine, aspartic acid, glutamic acid, glutamine, glycine, proline, and taurine.

[0083] Suitable inorganic acids can be selected from the following inorganic acids, alone or in combination: hydrochloric acid, sulfuric acid, and nitric acid. Hydrochloric acid is preferred as the acid for neutralization.

[0084] The acid can have a concentration from about 0.001M to the highest possible acid concentration. For example, the typical highest concentration for sulfuric acid is about 98% sulfuric acid. The acid can have a concentration from about 0.01M to 5M, 0.01M to 3M, or 0.1M to 2M. Preferably, the acid has a concentration of about 1M. The concentration of the acid can be about 0.01M, 0.05M, 0.10M, 0.15M, 0.20M, 0.25M, 0.30M, 0.35M, 0.40M, 0.45M, 0.50M, 0.55M, 0.60M, 0.65M, 0.70M, 0.75M, 0.80M, 0.85M, 0.90M, 0.95M, or 1.0M.

[0085] The acid may be present as an acidic solution comprising the acid and a non-solvent. The non-solvent may be any solvent in which chitosan is insoluble. Common non-solvents include ethyl lactate, ethyl acetate, methyl acetate, ethanol, acetone, or mixtures thereof. Preferred non-solvents include ethyl acetate or ethanol. More preferably, a non-solvent comprises 80:20 ethanol / water. It has been advantageously observed that the reaction proceeds at a faster rate when using a non-solvent comprising a mixture of 80:20 ethanol and water.

[0086] The ratio of chitosan to acid solution can be from about 5:1 to about 1:5. The ratio of chitosan to acid solution is preferably about 2:1.

[0087] In some embodiments, low-endotoxin basic chitosan can be mixed with acid for about 5 minutes. The reaction is then allowed to occur while the mixture is drying.

[0088] The solution obtained from the mixture of basic chitosan and acid may contain an acid salt. It is preferable to choose an alkaline solution and an acid to ensure that the resulting acid salt is biocompatible. For example, the alkaline solution may contain sodium hydroxide, and the acid may include hydrochloric acid. In this example, the acid salt is the biocompatible salt sodium chloride.

[0089] Acid salts are formed as byproducts of the reaction between basic chitosan and acid.

[0090] The presence of acid salts in the product can affect the effectiveness of the resulting chitosan product. For example, chitosan gels to a lesser extent in saline solution than in water, and even less in a double-concentration saline solution. The double-concentration saline solution mentioned in this article is considered to have 1.8% sodium chloride. Therefore, it is desirable to have the lowest possible amount of acid salts in the resulting chitosan product; ideally, the level of acid salts should have little or no difference in the effectiveness of the chitosan product.

[0091] It has been unexpectedly discovered that using an alkaline solution with a concentration of about 0.01 M to about 0.1 M produces the desired low endotoxin concentration while also generating fewer acid salt byproducts in subsequent processes, resulting in neutral chitosan, chitosan salts, or chitosan derivatives. Advantageously, fewer acid salt byproducts mean that the resulting chitosan product will have improved gelling properties in use compared to products containing higher amounts of acid salts. The method of the present invention can provide a chitosan product with suitable low amounts of acid salts without the need for washing or rinsing the chitosan product. This also has the additional advantage of eliminating the need for endotoxin-free water in washing or rinsing steps.

[0092] It has also been found that when preparing neutral chitosan, chitosan salts, or chitosan derivatives, the use of the low-concentration alkaline solution described herein results in minimal reduction in chitosan viscosity. Low-concentration alkalinity refers to about 0.01 M to about 1 M, preferably less than 1 M, more preferably about 0.02 M to about 0.2 M. In some embodiments, the alkalinity concentration can be 0.05 M. In some embodiments, the alkalinity concentration can be as described above. Therefore, it is advantageous that the use of a low-concentration alkaline solution in the method causes minimal damage to chitosan. Thus, endotoxins can be removed from chitosan while producing only minimal viscosity change. It is desirable that the chitosan viscosity decreases by less than about 25% during the process, preferably less than about 15%, more preferably less than about 10%.

[0093] When the method provides low-endotoxin neutral chitosan, the product is suitable as a starting material for preparing other chitosan-based products. One specific use is in the preparation of chitosan salts, whose absorption properties make them ideal for use as hemostatic agents to control bleeding. Preferably, the chitosan salts are water-soluble.

[0094] Therefore, in another embodiment of the invention, a low-endotoxin chitosan salt can be prepared by contacting the low-endotoxin neutral chitosan prepared by the method described herein with an acid.

[0095] The acid can be any acid suitable for providing the desired chitosan salt. For example, if chitosan acetate is required, acetic acid can be used; if chitosan succinate is required, succinic acid can be used, etc. Low endotoxin chitosan salts can be prepared in the method of this invention using any of the acids described herein.

[0096] The method for preparing low-endotoxin chitosan salts or chitosan derivatives may further include the step of drying a mixture of low-endotoxin neutral chitosan and acid. The drying step can be carried out by any conventional drying method known in the art. Preferably, the drying step is carried out in an oven or by filtering the product through an air dryer.

[0097] Therefore, the present invention provides low endotoxin neutral chitosan, chitosan salt or chitosan derivative having an endotoxin concentration of less than 100 EU / g.

[0098] Low endotoxin neutral chitosan can be water-insoluble.

[0099] Low endotoxin chitosan salts can be water-soluble.

[0100] According to another aspect of the invention, low endotoxin neutral chitosan, chitosan salt, or chitosan derivative is provided that can be obtained by any of the methods described herein.

[0101] According to another aspect of the invention, a neutral chitosan, chitosan salt, or chitosan derivative having an endotoxin concentration of less than 100 EU / g is provided.

[0102] Neutral chitosan, chitosan salt, or chitosan derivative preferably has an endotoxin concentration of less than 50 EU / g, more preferably less than 20 EU / g, even more preferably less than 15 EU / g, and most preferably less than 10 EU / g.

[0103] The low endotoxin chitosan salt of the present invention is suitable for use as a hemostatic agent to stop blood flow.

[0104] Therefore, according to another aspect of the invention, a low-endotoxin chitosan salt described herein is provided for use as a hemostatic agent to stop blood flow.

[0105] The low endotoxin chitosan salt of the present invention can be added to wound dressings for non-life-threatening or life-threatening surface bleeding.

[0106] Therefore, according to another aspect of the invention, a low endotoxin chitosan salt as described herein is provided for use in wound dressings for non-life-threatening or life-threatening bleeding.

[0107] The low-endotoxin chitosan salt of the present invention is suitable for preparing hemostatic wound dressings for stopping blood flow. According to another aspect of the invention, a hemostatic wound dressing comprising the low-endotoxin chitosan salt described herein is provided.

[0108] According to another aspect of the invention, a hemostatic material comprising the low endotoxin chitosan salt described herein is provided.

[0109] Hemostatic materials and / or chitosan salts can be in any suitable form, such as microparticles, powders, granules, flakes, fibers, gels, foams, sheets, films, or liquids.

[0110] According to another aspect of the invention, a method for stopping blood flow is provided, the method comprising the steps of: optionally, where possible, cleaning the wound area; applying a hemostatic wound dressing comprising a low-endotoxin chitosan salt described herein to the wound area; and applying constant pressure to the wound area until a gel block coagulates.

[0111] It is preferable to apply constant pressure to the wound area for about 3 minutes or longer. Attached Figure Description

[0112] Now, embodiments of the invention will be further described with reference to the accompanying drawings in the following non-limiting examples, wherein:

[0113] Figure 1 A graph showing the effect of different concentrations of acid salt byproducts on the viscosity of chitosan products in different media;

[0114] Figure 2 A graph showing the effect of acid treatment on the viscosity of chitosan polymers;

[0115] Figure 3 A diagram illustrating saline permeation in two different hemostatic materials of the present invention;

[0116] Figure 4 A graph showing the blood clotting time of two different hemostatic materials of the present invention;

[0117] Figure 5 A graph showing the percentage of hemostasis in an upper abdominal cutting test using two different hemostatic materials of the present invention. Detailed Implementation

[0118] Endotoxin test

[0119] 1. Prepare the USP (United States Pharmacopeia) extraction solution (4.6 ml 1M HCl and 45.4 ml endotoxin-free water) as described in the USP (United States Pharmacopeia) description of the chitosan endotoxin test;

[0120] 2. Extract by adding 0.1 g of the test chitosan product to 9.9 ml of USP extraction solution and maintaining at 37°C for 48 hours;

[0121] 3. After 48 hours, dilute 100 µl of the extract in 0.9 ml of endotoxin-free water; and

[0122] 4. Mix more than 100 µl of the substance into 100 µl of endotoxin-specific (ES) buffer provided by Charles River.

[0123] Endosafe®-PTS utilizes FDA-approved single-use columns. TM The extract was tested using a handheld spectrophotometer. The extraction process used a 2000x dilution and a minimum detection limit of 10 EU / g. Example

[0124] Example 1:

[0125] Mix 50g of chitosan (Primex Iceland) with 50g of 1M NaOH for 30 minutes. Keep the resulting moist alkaline chitosan chips at room temperature for 48 hours. Then dry them in an oven on a tray at 40°C.

[0126] Initial endotoxin level of raw chitosan: 383 EU / g

[0127] Dry-processed alkaline chitosan: 10.6 EU / g

[0128] Control (0.1g of endotoxin-free water used in the extraction process instead of chitosan): <10 EU / g

[0129] Example 2:

[0130] Mix 50g of chitosan (Primex Iceland) with 50g of 0.5M NaOH for 10 minutes. Keep the resulting moist alkaline chitosan chips at room temperature for 72 hours. Then dry them in an oven on a tray at 40°C.

[0131] Initial endotoxin level of raw chitosan: 383 EU / g

[0132] Dry-processed alkaline chitosan: 38.3 EU / g

[0133] Example 3:

[0134] Mix 50g of chitosan (Primex Iceland) with 50g of 0.2M NaOH for 10 minutes. Keep the resulting moist alkaline chitosan fragments at room temperature for 7 days. Then dry them in an oven on a tray at 40°C.

[0135] Initial endotoxin level of raw chitosan: 383 EU / g

[0136] Dry-processed alkaline chitosan: 27.9 EU / g

[0137] Example 4:

[0138] Mix 50g of chitosan (Primex Iceland) with 50g of 0.1M NaOH for 10 minutes. Keep the resulting moist alkaline chitosan fragments at room temperature for 14 days. Then dry them in an oven on a tray at 40°C.

[0139] Initial endotoxin level of raw chitosan: 383 EU / g

[0140] Dry-processed alkaline chitosan: 12.7 EU / g

[0141] Example 5:

[0142] Mix 50g of chitosan (Primex Iceland) with 100g of 0.2M NaOH for 10 minutes. Keep the resulting moist alkaline chitosan fragments at room temperature for 2 days. Then dry them in an oven on a tray at 40°C.

[0143] Initial endotoxin level of raw chitosan: 383 EU / g

[0144] Dry-processed alkaline chitosan: 35.7 EU / g

[0145] Example 6:

[0146] Mix 50g of chitosan (Primex Iceland) with 100g of 0.1M NaOH for 10 minutes. Keep the resulting moist alkaline chitosan fragments at room temperature for 2 days. Then dry them in an oven on a tray at 40°C.

[0147] Initial endotoxin level of raw chitosan: 383 EU / g

[0148] Dry-processed alkaline chitosan: 49.3 EU / g

[0149] Example 7:

[0150] Mix 50g of chitosan powder (Cognis, Germany) with 50g of 0.1M NaOH for 10 minutes. Keep the resulting moist alkaline chitosan fragments at room temperature for 7 days. Then dry them in an oven on a tray at 40°C.

[0151] Initial endotoxin level of raw chitosan: 45.3 EU / g

[0152] Dry-treated alkaline chitosan: <10 EU / g

[0153] Control (0.1g of endotoxin-free water used in the extraction process instead of chitosan): <10 EU / g

[0154] Example 8:

[0155] Mix 50g of chitosan powder (Cognis, Germany) with 50g of 0.05M NaOH for 10 minutes. Keep the resulting moist alkaline chitosan fragments at room temperature for 7 days. Then dry them in an oven on a tray at 40°C.

[0156] Initial endotoxin level of raw chitosan: 45.3 EU / g

[0157] Dry-treated alkaline chitosan: <10 EU / g

[0158] Control (0.1g of endotoxin-free water used in the extraction process instead of chitosan): <10 EU / g

[0159] Example 9:

[0160] Mix 50g of chitosan powder (Cognis, Germany) with 50g of 0.025M NaOH for 10 minutes. Keep the resulting moist alkaline chitosan fragments at room temperature for 7 days. Then dry them in an oven on a tray at 40°C.

[0161] Initial endotoxin level of raw chitosan: 45.3 EU / g

[0162] Dry-processed alkaline chitosan: <13.5 EU / g

[0163] Control (0.1g of endotoxin-free water used in the extraction process instead of chitosan): <10 EU / g

[0164] This process can also be scaled up and used to prepare larger batches. Examples 10 and 11 were prepared in a Class 100,000 cleanroom (US FED STD 209E cleanroom standard) commonly used in medical device manufacturing.

[0165] Example 10:

[0166] Mix 3.5 kg of chitosan powder (Primex Iceland) with 3.5 kg of 0.1 M NaOH for 30 minutes. Keep the resulting moist alkaline chitosan fragments at room temperature for 14 days. Then dry at 40 °C by filtration through an air dryer.

[0167] Initial endotoxin level of raw chitosan: 288 EU / g

[0168] Dry-processed alkaline chitosan: 10.2 EU / g

[0169] Control (0.1g of endotoxin-free water used in the extraction process instead of chitosan): <10 EU / g

[0170] Example 11:

[0171] Mix 3.5 kg of chitosan (Primex Iceland) powder with 3.5 kg of 1 M NaOH for 30 minutes. Keep the resulting moist alkaline chitosan fragments at room temperature for 24 hours. Then dry at 40°C by filtration through an air dryer.

[0172] Initial endotoxin level of raw chitosan: 288 EU / g

[0173] Dry-processed alkaline chitosan: 15.3 EU / g

[0174] Control (0.1g of endotoxin-free water used in the extraction process instead of chitosan): <10 EU / g

[0175] This process can be applied to chitosan in different physical forms, such as chitosan fibers or chitosan fabrics.

[0176] Example 12:

[0177] Mix 10g of chitosan fiber (1.8dtex x 28mm) with 10g of 0.1M NaOH for 10 minutes. Keep the resulting moist alkaline chitosan fiber at room temperature for 2 days. Then dry it on a tray at 40°C in a laboratory oven.

[0178] Initial endotoxin level of raw material chitosan fiber: 88 EU / g

[0179] Dry-treated alkaline chitosan fibers: <10 EU / g

[0180] Control (0.1g of endotoxin-free water used in the extraction process instead of chitosan): <10 EU / g

[0181] Example 13:

[0182] Mix 5g of chitosan nonwoven fabric (60g sm) with 5g of 0.1M NaOH for 10 minutes. Keep the resulting wet alkaline chitosan fabric at room temperature for 2 days. Then dry it on a tray in a laboratory oven at 40°C.

[0183] Initial endotoxin level in raw material chitosan fabric: 401 EU / g

[0184] Dry-treated alkaline chitosan fabric: <78 EU / g

[0185] Control (0.1g of endotoxin-free water used in the extraction process instead of chitosan): <10 EU / g

[0186] Examples 14 and 15 below utilize a base different from sodium hydroxide.

[0187] Example 14:

[0188] Mix 50g of chitosan (Primex Iceland) with 50g of 0.2M KOH (potassium hydroxide) for 30 minutes. Keep the resulting moist alkaline chitosan fragments at room temperature for 7 days. Then dry them on a tray in a laboratory oven at 40°C.

[0189] Initial endotoxin level of raw chitosan: 383 EU / g

[0190] Dry-processed alkaline chitosan: 14.4 EU / g

[0191] Control (0.1g of endotoxin-free water used in the extraction process instead of chitosan): <10 EU / g

[0192] Example 15:

[0193] Mix 50g of chitosan (Primex Iceland) with 50g of 0.5M sodium carbonate for 30 minutes. Keep the resulting moist alkaline chitosan chips at room temperature for 7 days. Then dry them on a tray in a laboratory oven at 40°C.

[0194] Initial endotoxin level of raw chitosan: 383 EU / g

[0195] Dry-processed alkaline chitosan: 25.8 EU / g

[0196] Control (0.1g of endotoxin-free water used in the extraction process instead of chitosan): <10 EU / g.

[0197] Examples 1-15 all relate to the preparation of low-endotoxin basic chitosan. This low-endotoxin basic chitosan can then be used as a raw material to prepare other chitosan-based products. For example, the basic chitosan can be neutralized to pH 7 by adding a low level of a suitable acid that reacts with an alkali to produce a biocompatible salt, thus generating neutral chitosan. For example, if sodium hydroxide is used in the alkaline solution, neutralization can be achieved by adding hydrochloric acid. The product will contain a low amount of residual sodium chloride.

[0198] Example 16:

[0199] 20 g of wet alkaline chitosan fragments from Example 4 were weighed into a beaker. This contained 10 g of chitosan and 10 g of 0.1 M NaOH. To neutralize the NaOH, 1 g of 1 M HCl was required. This was mixed with 9 g of ethanol in a separate beaker. The acid solution was then incorporated into the wet alkaline chitosan fragments and stirred for 5 minutes. The resulting mixture was then dried in a laboratory oven at 40 °C. It contained 0.29% sodium chloride.

[0200] Example 17:

[0201] 20 g of wet alkaline chitosan fragments from Example 4 were weighed into a beaker. This contained 10 g of chitosan and 10 g of 0.1 M NaOH. To neutralize the NaOH, 1 g of 1 M acetic acid was required. This was mixed with 9 g of ethanol in a separate beaker. The acid solution was then incorporated into the wet alkaline chitosan fragments and stirred for 5 minutes. The resulting mixture was then dried in a laboratory oven at 40 °C. It contained 0.5% sodium acetate.

[0202] The low-endotoxin basic chitosan generated in Examples 1-15 can also be used to prepare low-endotoxin water-soluble chitosan salts or other chitosan derivatives. Advantageously, this can be achieved without requiring a sterile environment, large quantities of expensive endotoxin-free water, or rinsing or washing. For example, the low-endotoxin basic chitosan can be reacted with a larger level of suitable acid. A small portion of the acid will react with the base to produce a biocompatible salt.

[0203] Example 18:

[0204] In a Class 100,000 cleanroom, 3.1 kg of dried, low-endotoxin, alkaline chitosan from Example 10 was weighed into a stainless steel mixer. 3.6 kg of lactic acid was premixed with 0.9 kg of endotoxin-free water. This mixture was sprayed onto the chitosan while the mixer was running. The resulting material was dried at 40°C by filtration through an air dryer.

[0205] The material was found to be completely water-soluble.

[0206] Initial endotoxin level of raw chitosan: 288 EU / g

[0207] Dry-treated alkaline chitosan: 10.2 EU / g (Example 10)

[0208] Dry water-soluble chitosan: 13.8 EU / g

[0209] Control (0.1g of endotoxin-free water used in the extraction process instead of chitosan): <10 EU / g

[0210] Example 19:

[0211] In a Class 100,000 cleanroom, 3.1 kg of dried, low-endotoxin, alkaline chitosan from Example 11 was weighed into a stainless steel mixer. 3.9 kg of lactic acid was premixed with 0.9 kg of endotoxin-free water. This mixture was sprayed onto the chitosan while the mixer was running. The resulting material was dried at 40°C by filtration through an air dryer.

[0212] The material was found to be completely water-soluble.

[0213] Initial endotoxin level of raw chitosan: 288 EU / g

[0214] Dry-treated alkaline chitosan: 15.3 EU / g (Example 4)

[0215] Dry water-soluble chitosan: 14.6 EU / g

[0216] Control (0.1g of endotoxin-free water used in the extraction process instead of chitosan): <10 EU / g

[0217] In another embodiment, low-endotoxin basic chitosan can also be used as a raw material to prepare low-endotoxin chitosan derivatives, such as carboxymethyl chitosan.

[0218] Example 20:

[0219] 20 g of wet alkaline chitosan fragments from Example 1 were weighed into a beaker. This contained 10 g of chitosan and 10 g of 1 M NaOH. In a separate beaker, a mixture of 5 g of sodium chloroacetate, 5 g of water, and 10 g of ethanol was prepared. The mixture was then stirred into the wet alkaline chitosan fragments. The temperature was then raised to 60 °C and maintained for 4 hours. Before drying in a laboratory oven at 40 °C, the resulting mixture was washed three times with 10 g of ethanol to remove any residual sodium chloroacetate.

[0220] Effect of acid salts on viscosity:

[0221] Low-endotoxin basic chitosan reacts with acid to produce neutral pH chitosan or chitosan salts, generating acidic salt byproducts. The presence of these byproducts can affect the properties of the chitosan product. For example, the level of these byproducts can affect the viscosity of the chitosan product in brine.

[0222] refer to Figure 1 The results show the effects of adding sodium lactate to saline solution at different concentrations and its influence on the viscosity of a 2g sample of the currently commercially available chitosan product CELOX® after 3 minutes in 20g solutions in different media.

[0223] The base medium is saline solution derived from bodily fluids, to which different levels of sodium lactate are added. Sodium lactate represents a byproduct of the reaction between sodium hydroxide and lactic acid.

[0224] The results are shown in Table 1 and Figure 1 middle.

[0225] Table 1

[0226]

[0227] from Figure 1 It is clearly observed that the viscosity of CELOX® in the medium decreases as the added salt level increases. Therefore, advantageously, the chitosan product of this invention has only a low level of residual salt byproducts.

[0228] Effect of low concentration alkaline solutions on viscosity:

[0229] The low-endotoxin basic chitosan prepared in Example 10 was tested to demonstrate the effect of acid treatment on the viscosity of the chitosan polymer, which is considered a measure of molecular weight. The experiment was conducted according to the following steps:

[0230] a) Weigh out 5g of the low-endotoxin basic chitosan particles prepared in Example 10;

[0231] b) Weigh 4.95g of acetic acid into a 600ml beaker;

[0232] c) Add 490.05g of deionized water to a beaker to prepare 495g of 1% acetic acid solution;

[0233] d) Place the beaker on the stirrer pan and start stirring (increase the stirring speed as the solution viscosity increases);

[0234] e) Add chitosan particles to the acetic acid solution;

[0235] f) Check the solution frequently until all particles have dissolved, and increase the stirring level as the solution viscosity increases, if necessary;

[0236] g) Measured when introducing chitosan particles into the acetic acid solution, with the solution held for a total of 24 hours;

[0237] h) Connect rotor 64 to the Brookfield viscometer

[0238] i) Set the rotor to 10 rpm;

[0239] j) Insert the rotor into the solution onto the mark on the rotor, turn on the viscometer, and allow it to stabilize;

[0240] k) Record viscosity (cPs) at selected time intervals.

[0241] The results of the above viscosity tests are shown in Table 2 below. Figure 2 In the middle. For each batch, the three readings are averaged at each time interval.

[0242] Table 2

[0243]

[0244] Available from Figure 2 The viscosity measurements shown suggest that the molecular weight of the low-endotoxin basic chitosan polymer prepared with 0.1M sodium hydroxide solution is stable for several weeks.

[0245] Effect of decreasing concentration of alkaline solutions

[0246] The effect of using a low concentration of alkaline solution in the method of the present invention was tested in three experiments, focusing on (1) the percentage of saline solution permeability into the test sample; (2) the time for blood clotting; and (3) the percentage of hemostasis in an in vivo model of an upper abdominal incision.

[0247] refer to Figure 3 The results of the penetration test are shown in the image.

[0248] The general test method is as follows: Add 5 ml of distilled water to a test tube. Add 1 drop of red food dye to the water. Gently pour 3 g of the sample hemostatic powder onto the surface of the water to allow a layer to form. After 1 minute, measure the distance the water travels into the hemostatic powder and record it as the percentage of penetration.

[0249] Available Figure 3 It was observed that, compared with the hemostatic powder prepared with 0.1M sodium hydroxide, the hemostatic powder prepared with 1.0M sodium hydroxide was less stable over time. The increased penetration of saline solution into the particles indicated that blood had a greater chance of passing through the particles without forming a gel plug.

[0250] refer to Figure 4 The results of the blood clotting test are displayed.

[0251] The general experimental method is as follows: Add 0.75g of the sample hemostatic powder to a test tube, and then add 5ml of heparinized rabbit blood. Invert the test tube and record the time it takes for the blood to completely coagulate into a gel block.

[0252] Available Figure 4 It was observed that the hemostatic powder prepared with 1.0M sodium hydroxide took longer to clot blood than the hemostatic powder prepared with 0.1M sodium hydroxide. The time was approximately three times longer, which relates to lower hemostatic properties (data have shown that time periods longer than 180 seconds do not result in 100% hemostasis in low-pressure, medium-volume internal bleeding models).

[0253] refer to Figure 5 The results of the hemostasis test are displayed.

[0254] The general experimental method is as follows: A 3-5 cm incision is made in the upper abdominal artery of a pig model (non-heparinized). Granular hemostatic material is applied, and pressure is applied for 1 minute. If rebleeding occurs, pressure is applied for an additional minute.

[0255] Available Figure 5 It was observed that, compared to 100% hemostatic granules prepared with 0.1M sodium hydroxide, hemostatic granules prepared with 1.0M sodium hydroxide achieved 60% experimental hemostasis.

[0256] The experimental results show that the lower the concentration of the alkaline solution used in preparing the hemostatic material of the present invention, the better the material's permeability, blood coagulation, and hemostasis performance.

[0257] Of course, it should be understood that the present invention is not intended to be limited to the above embodiments, which are described only as examples.

Claims

1. A method for preparing low-endotoxin basic chitosan, the method comprising the following steps: (a) Contacting chitosan with an alkaline solution of 0.01M-0.2M concentration to form a mixture; and (b) Allow the mixture to remain for at least 12 hours. In the method described above, the viscosity of chitosan is reduced by less than 25%.

2. The method of claim 1, wherein the method further comprises the step (c) of drying the mixture.

3. The method of claim 1 or 2, wherein the mixture is kept for 24 hours in step (b).

4. The method of any of the preceding claims, wherein the concentration of the alkaline solution is from 0.02M to 0.2M.

5. The method of claim 4, wherein the concentration of the alkaline solution is 0.1 M.

6. The method of any of the preceding claims, wherein the alkaline solution and chitosan are present in a ratio of 1 part chitosan to 10 parts alkaline solution to a ratio of 10 parts chitosan to 1 part alkaline solution.

7. The method of any of the preceding claims, wherein the alkaline solution comprises an alkali or alkaline earth metal component selected from the following components alone or in combination: metal hydroxides, metal carbonates, metal bisulfites, metal persilicates, conjugate bases, and ammonium hydroxides.

8. The method of claim 7, wherein the metal is selected from sodium, potassium, calcium or magnesium.

9. The method of claim 7 or 8, wherein the alkaline component is selected from sodium hydroxide, potassium hydroxide, or sodium carbonate.

10. The method of any of the preceding claims, wherein an alkaline solution is sprayed onto chitosan, or chitosan is mixed with an alkaline solution.

11. The method of any of the preceding claims, wherein the mixture is kept for at least 48 hours in step (b).

12. The method of claim 11, wherein the mixture is kept for 2 to 4 weeks.

13. The method of any of the preceding claims, wherein the mixture is kept in a clean container and / or kept under an inert atmosphere.

14. The method of any of the preceding claims, wherein the mixture further comprises a preservative.

15. The method of claim 14, wherein the preservative is selected from silver ions, zinc ions, chlorhexidine, or combinations thereof.

16. The method of any of the preceding claims, wherein the drying step is performed in an oven.

17. A method for preparing low endotoxin neutral chitosan, chitosan salt, or chitosan derivative, the method comprising the step of contacting an acid with an alkaline chitosan prepared by any one of claims 1 to 16.

18. The method of claim 17, wherein the step of contacting the basic chitosan with the acid is performed prior to the drying step (c) of any one of claims 2 to 16.

19. The method of claim 17 or 18, wherein the acid is sprayed onto the basic chitosan, or the basic chitosan is mixed with the acid.

20. The method of any one of claims 17 to 19, wherein the acid is a Lewis acid.

21. The method of any one of claims 17 to 19, wherein the acid is selected from the following acids, alone or in combination: organic acids and inorganic acids.

22. The method of any one of claims 17 to 19, wherein the acid is selected from the following acids, alone or in combination: carboxylic acids and nucleic acids.

23. The method of any one of claims 17 to 19, wherein the acid is selected from the following acids, alone or in combination: fatty acids and amino acids.

24. The method of any one of claims 17 to 19, wherein the acid is selected from the following acids, alone or in combination: monoprotic acids and diprotic acids.

25. The method of any one of claims 17 to 19, wherein the acid is a polyprotic acid.

26. The method of claim 21, wherein the organic acid is selected from the following organic acids, alone or in combination: acetic acid, tartaric acid, citric acid, ascorbic acid, acetylsalicylic acid, gluconic acid, and lactic acid.

27. The method of claim 23, wherein the fatty acid is selected from the following fatty acids, alone or in combination: myristone acid, palmitoleic acid, cis-6-hexadecenoic acid, oleic acid, trans-oleic acid, 11-octadecenoic acid, linoleic acid, trans-linoleic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, docosahexaenoic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, benzyl acid, ceric acid, and ceric acid.

28. The method of claim 23, wherein the amino acid is selected from the following amino acids, alone or in combination: histidine, lysine, aspartic acid, glutamic acid, glutamine, glycine, proline, and taurine.

29. The method of claim 21, wherein the inorganic acid is selected from the following inorganic acids, alone or in combination: hydrochloric acid, sulfuric acid, and nitric acid.

30. The method of any one of claims 17 to 29, wherein the acid has a concentration of 1 M.

31. The method of any one of claims 17 to 30, wherein the acid is present as an acid solution, the acid solution comprising an acid and a non-solvent.

32. The method of claim 31, wherein the non-solvent is selected from ethyl lactate, ethyl acetate, methyl acetate, ethanol, acetone, an 80:20 mixture of ethanol and water, or a mixture thereof.

33. The method of claim 31 or 32, wherein the ratio of chitosan to acid is 5:1 to 1:

5.

34. The method of any one of claims 17 to 33, wherein the basic chitosan is mixed with the acid for 5 minutes.

35. The method of any one of claims 17 to 34, the method further comprising the step of drying the reaction product.

36. The method of claim 35, wherein the drying step is carried out in an oven or by filtering the product through an air dryer.

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